Resistance to HER2-targeted therapies in HER2-positive breast cancer

Breast cancer accounts for 522,000 deaths worldwide each year and is the most common cancer in women. It is classified according to the cell of origin and its expression of several receptors: oestrogen and progesterone receptors, and human epidermal growth factor receptor 2 (HER2). Historically, HER2-positive breast cancers had a worse survival prognosis than oestrogen or progesterone receptor-positive cancers, but the development of HER2-targeted therapies led to significant survival improvements. Despite this, patients often present with de novo resistance, or will develop acquired resistance to targeted therapies. Several resistance mechanisms have been identified but attempts to target them have failed. Thus, it is of paramount importance to identify the mechanisms used, to prevent development of resistance or resensitise tumours to HER2-targeted therapies. Objectives of this study were: to understand the link between epithelial to mesenchymal transition (EMT) and loss of HER2, seen in a model of acquired resistance to the HER2-targeted therapy, sapatinib, and to characterise and validate tumours from a sapatinib-treated spontaneous mouse model of HER2-positive breast cancer. The EMT-linked transcription factor ZEB1 was associated with acquired resistance to sapatinib in tumours that had undergone EMT and concurrently lost HER2. Generation of drug resistant cell lines failed to recapitulate the in vivo phenotype. Transient overexpression of ZEB1 in vitro did not induce clear EMT or loss of HER2, despite a trend towards lower HER2 expression. However, we found that treatment of cells with ERBB2 shRNA, the gene encoding HER2, increased levels of ZEB1 and enhanced migration, but did not induce overt EMT. This may be the result of differing PTEN status between in vivo and in vitro models. Treatment of a spontaneous mouse model of HER2-positive breast cancer with sapatinib revealed that progressing tumours had an increase in proteins associated with cellular iron homeostasis. Further investigation revealed increased heme oxygenase-1 (HO-1), iron exporter ferroportin and altered iron storage. To ascertain if modulation of dietary iron intake could affect the development of resistance to sapatinib, mice were given a control or iron-deficient diet and treated with vehicle or sapatinib. This showed that in sapatinib-treated mice fed an iron-deficient diet, HO-1 was not increased as in tumours from mice fed a iron-low control diet. We looked at the possibility of HER2-targeting therapies inducing ferroptosis, an iron-dependent form of cell death. Sapatinib-treated tumours from mice on a iron-low control diet had increased cyclooxygenase 2 (COX2), a marker of ferroptosis, which was not seen in sapatinib-treated tumours from mice on an iron-deficient diet. Additionally, in vitro drug treatments with HER2-targeting agents showed that SKBR3 cell death could be rescued by iron chelation. HO-1 overexpression in SKBR3 cells revealed increased autophagic flux and resistance to HER2-targeted therapies. Inhibition of autophagy reversed resistance, rendering them susceptible to sapatinib- and lapatinib-induced cell death. Further, increased autophagic flux was seen in all progressive tumours on sapatinib. The increased resistance to sapatinib in mice fed an iron-deficient diet was also associated with increased autophagic flux, although this was HO-1-independent. Taken together, the results presented here provide a novel mechanism of cell death induced by HER2-targeting agents in vitro and in vivo. We have shown that increased HO-1 and reducing dietary iron can affect the development of resistance to sapatinib, which is reliant on autophagy induction. Further, inhibiting autophagy can resensitise cells to sapatinib and lapatinib treatment

Proangiogenic contribution of adiponectin toward mammary tumor growth in vivo

PURPOSE: Adipocytes represent one of the most abundant constituents of the mammary gland. They are essential for mammary tumor growth and survival. Metabolically, one of the more important fat-derived factors (“adipokines”) is adiponectin (APN). Serum concentrations of APN negatively correlate with body mass index and insulin resistance. To explore the association of APN with breast cancer and tumor angiogenesis, we took an in vivo approach aiming to study its role in the mouse mammary tumor virus (MMTV)-polyoma middle T antigen (PyMT) mammary tumor model. EXPERIMENTAL DESIGN: We compared the rates of tumor growth in MMTV-PyMT mice in wild-type and APN-null backgrounds. RESULTS: Histology and micro-positron emission tomography imaging show that the rate of tumor growth is significantly reduced in the absence of APN at early stages. PyMT/APN knockout mice exhibit a reduction in their angiogenic profile resulting in nutrient deprivation of the tumors and tumor-associated cell death. Surprisingly, in more advanced malignant stages of the disease, tumor growth develops more aggressively in mice lacking APN, giving rise to a larger tumor burden, an increase in the mobilization of circulating endothelial progenitor cells, and a gene expression fingerprint indicative of more aggressive tumor cells. CONCLUSIONS: These observations highlight a novel important contribution of APN in mammary tumor development and angiogenesis, indicating that APN has potent angio-mimetic properties in tumor vascularization. However, in tumors deprived of APN, this antiangiogenic stress results in an adaptive response that fuels tumor growth through mobilization of circulating endothelial progenitor cells and the development of mechanisms enabling massive cell proliferation despite a chronically hypoxic micro-environment

A Distinct Macrophage Population Mediates Metastatic Breast Cancer Cell Extravasation, Establishment and Growth

Background: The stromal microenvironment and particularly the macrophage component of primary tumors influence their malignant potential. However, at the metastatic site the role of these cells and their mechanism of actions for establishment and growth of metastases remain largely unknown. Methodology/Principal Findings: Using animal models of breast cancer metastasis, we show that a population of host macrophages displaying a distinct phenotype is recruited to extravasating pulmonary metastatic cells regardless of species of origin. Ablation of this macrophage population through three independent means (genetic and chemical) showed that these macrophages are required for efficient metastatic seeding and growth. Importantly, even after metastatic growth is established, ablation of this macrophage population inhibited subsequent growth. Furthermore, imaging of intact lungs revealed that macrophages are required for efficient tumor cell extravasation. Conclusion/Significance: These data indicate a direct enhancement of metastatic growth by macrophages through their effects on tumor cell extravasation, survival and subsequent growth and identifies these cells as a new therapeutic target fo

Loss of retinal cadherin facilitates mammary tumor progression and metastasis

The mammary epithelium is thought to be stabilized by cellcell adhesion mediated mainly by E-cadherin (E-cad). Here, we show that another cadherin, retinal cadherin (R-cad), is critical for maintenance of the epithelial phenotype. R-cad is expressed in nontransformed mammary epithelium but absent from tumorigenic cell lines. In vivo, R-cad was prominently expressed in the epithelium of both ducts and lobules. In human breast cancer, R-cad was down-regulated with tumor progression, with high expression in ductal carcinoma in situ and reduced expression in invasive duct carcinomas. By comparison, E-cad expression persisted in invasive breast tumors and cell lines where R-cad was lost. Consistent with these findings, R-cad knockdown in normal mammary epithelium stimulated invasiveness and disrupted formation of acini despite continued E-cad expression. Conversely, R-cad overexpression in aggressive cell lines induced glandular morphogenesis and inhibited invasiveness, tumor formation, and lung colonization. R-cad also suppressed the matrix metalloproteinase 1 (MMP1), MMP2, and cyclooxygenase 2 gene expression associated with pulmonary metastasis. The data suggest that R-cad is an adhesion molecule of the mammary epithelium, which acts as a critical regulator of the normal phenotype. As a result, R-cad loss contributes to epithelial suppression and metastatic progression. ©2009 American Association for Cancer Research.Fil: Agiostratidou, Georgia. Albert Einstein College of Medicine, NY; Estados UnidosFil: Li, Maomi. Albert Einstein College of Medicine, NY; Estados Unidos. Montefiore Medical Center, NY; Estados UnidosFil: Suyama, Kimita. Albert Einstein College of Medicine, NY; Estados UnidosFil: Badano, Ines. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Misiones. Facultad de Ciencias Exactas, Químicas y Naturales. Laboratorio de Biología Molecular Aplicada; Argentina. Albert Einstein College of Medicine, NY; Estados UnidosFil: Keren, Rinat. Albert Einstein College of Medicine, NY; Estados UnidosFil: Chung, Su. Albert Einstein College of Medicine, NY; Estados UnidosFil: Anzovino, Amy. Albert Einstein College of Medicine, NY; Estados UnidosFil: Hulit, James. Albert Einstein College of Medicine, NY; Estados UnidosFil: Qian, Binzhi. Albert Einstein College of Medicine, NY; Estados UnidosFil: Bouzahzah, Boumediene. Albert Einstein College of Medicine, NY; Estados UnidosFil: Eugenin, Eliseo. Albert Einstein College of Medicine, NY; Estados UnidosFil: Loudig, Olivier. Albert Einstein College of Medicine, NY; Estados UnidosFil: Phillips, Greg R.. Mount Sinai School of Medicine, NY; Estados UnidosFil: Locker, Joseph. Albert Einstein College of Medicine, NY; Estados UnidosFil: Hazan, Rachel B.. Albert Einstein College of Medicine, NY; Estados Unido